1. Field of the Invention
This invention relates to the field of the preservation of embryos at low temperatures; it more particularly relates to an improvement in the methods of preparation of equine embryos for cryopreservation (freezing or vitrification), especially for subsequent transplantation.
The freezing of embryos during embryo transfers has many advantages. Amongst others, it removes the need to synchronize the oestral cycles of the donor and the recipient; it simplifies the transport of embryos over long distances and it also makes the creation of embryo banks possible, especially for rare animals or endangered species.
2. Description of the Related Art
After the first successes in the 1970""s, embryo freezing has now become a routine technique in domestic ruminants, especially cattle.
Parallel work carried out in the equine field led to the first births of foals from frozen embryos at the beginning of the 1980""s.
Biologically, the fecundation of the equine embryo takes place in the oviduct, at the junction between the ampulla and the isthmus, generally 12 hours after ovulation. The embryo then develops in the oviduct; it passes from the unicellular zygotic stage, from 100 to 160 xcexcm in size, to the morula stage at from 16 to 32 cells (150 to 200 xcexcm). The mitotic divisions at first take place simultaneously in the different blastomeres, then more and more asynchronously. The development phase of the equine embryo in the oviduct lasts about 5 days.
The blastocyte stage follows the morula stage at about the point when the embryo passes into the uterus. It is at this stage that differentiation between two cell populations takes place: the trophoblast and the inner cell mass which becomes the foetus.
At this stage, the embryo is composed of a liquid cavity (the blastocoel), surrounded by a cellular layer (the trophoblast), next to the inner cell mass. Around the embryo are two acellular structures, the pellucid zone and the capsule.
The pellucid zone is of the order of 15 to 30 xcexcm thick; it is composed of a dense inner layer, with a small number of fine canals, and an outer layer containing large lacunae. This pellucid zone is present in the ovocyte and disappears around the 7th or 8th day.
The capsule is specific to equine embryos. It is formed in about 8 hours after the embryo enters the uterus, between the trophoblast and the pellucid zone. This capsule is composed of glycoproteins. Its thickness increases steadily up to a peak at about the 18th day, and then decreases until it disappears at around the 22nd day, at about the time when the embryo is fixed in the uterus.
Embryos intended for freezing must be collected before they are fixed in the uterus. The suitable time is evaluated, after covering or insemination, by transrectal palpation and from echotomographic data.
This collection may be made very soon after fecundation, in the oviduct, but this requires a surgical operation which is traumatic for the animal. The other solution is to wait until the embryo reaches the uterus (after the 6th day after fecundation) and to collect through the vagina, using a suitable collection probe, following the technique of LAGNEAUX D. et al. (1988, xe2x80x9cla transplantation embryonnaire chez la jumentxe2x80x9d, CEREOPA, 14th day, 163-181). This probe can be used to introduce a collection medium into the uterus (for example Dubelco""s phosphate buffered saline (PBS)+2 g/l of bovine serum albumin, available from I.M.V. (Instrument de Mxc3xa9decine Vxc3xa9txc3xa9rinaire), B.P. 81, 61 L""AIGLE (France)), then to recover it with the possible embryo(s). The recovery medium is examined with a binocular microscope and, if embryos are found, they are isolated in a small vessel containing the same medium (PBS+albumin).
The embryo is then prepared to support the subsequent freezing operation. This preparation consists of subjecting it to the action of cryoprotectants whose particular function is to prevent the formation of intracellular crystals during the reduction in temperature. The cryoprotectants used may be selected from glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, or 1,2-propanediol ; they are used in concentrations of the order of 1 to 2M, for a duration of 10 to 30 min (these concentrations and times are varied according to the protection efficiency desired, taking account of the possible toxicity of the products). The embryo is preferably subjected to successive baths of cryoprotectants in increasing concentration.
The embryo is then placed in a plastic straw with a small amount of the medium corresponding to the final bath of cryoprotectant, and this straw is then placed in the freezing compartment of a programmable freezer at a temperature of about xe2x88x927xc2x0 C. After an equilibration time of about 5 to 10 min, the crystallization of the contents is induced by contact with a metal rod previously cooled in liquid nitrogen (xe2x88x92196xc2x0 C.). After a further time for thermal equilibrium, the freezer programmer reduces the temperature to about xe2x88x9230xc2x0 C./xe2x88x9235xc2x0 C., at a rate of xe2x88x920.1 to 1xc2x0 C. per minute; at this temperature, the straw is immersed in liquid nitrogen, then stored in a tank.
Thawing, before transfer, is achieved by immersing the straw in a water-bath at 37xc2x0 C. for 1 min; the embryo is taken out and the operation of removing the cryoprotectant is performed by a succession of dilutions in baths of decreasing concentration.
The embryo is then ready for transfer into the recipient mare. Two techniques are used conventionally : the first, surgical, consists of implanting the embryo in the oviduct; the second, non-surgical and better corresponding to the objectives of commercial transfer, consists of placing the embryo in the uterus using a probe to pass through the cervix.
The technique described above is derived from that used for many years in cattle, and which in cattle gives a gestation rate of greater than 50% from frozen embryos.
However, in the equine field, the gestation rates obtained are very low, only of the order of 20 to 30%.
It has only recently been found that the use of this freezing technique in equines led to significant necroses in the inner cell mass and trophoblast of the embryo, and this seemed to be the cause of the high number of premature abortions.
The aim of the present invention is to provide an improved technique for preparing the embryos for cryopreservation, which leads to a reduced level of the necroses associated with this operation, and in consequence favourizes the subsequent gestation after the embryo transfer.
The method according to the present invention is based on the presence of the capsule which surrounds the embryo for the greater part of the time which the embryo spends in the uterus of the donor, and on the hypothesis that the presence of this capsule hinders the operations of preparing the embryo before freezing.
In order to achieve an acceptable collection level, the embryos are collected fairly late; as a general rule, they are at the blastocyte stage and surrounded by a capsule.
In the conventional sequence of operations for preparing equine embryos for cryopreservation, the method according to the invention consists of, prior to subjecting said embryo to the action of cryoprotectants, subjecting it to an appropriate treatment to eliminate the embryonic capsule or to increase the permeability of this capsule, in order to reinforce the subsequent action of said cryoprotectant(s).
According to a first embodiment, the capsule is destroyed or simply made more permeable by an appropriate chemical or enzymatic technique, for example based on collagenase or trypsin. The concentrations of the products used and the contact times (bath treatments) are selected so that the capsule is at least partially lysed while taking account of the possible toxicity of the products so as not to harm the embryo. Since the sites of action of trypsin and collagenase are different, the use of a mixture of these two enzymes may be envisaged.
According to another technique, the capsule is treated mechanically: it is partially or completely removed, or simply pierced. This mechanical treatment is advantageously preceded by a suitable chemical or enzymatic technique to weaken said capsule.